LncRNA MALAT1 functions as a biomarker of no-reflow phenomenon in ST-segment elevation myocardial infarction patients receiving primary percutaneous coronary intervention

MALAT1 was reported to sponge miR-30e, miR-126 and miR-155 in the pathogenesis of many diseases. Plasma miR-30e can indicate the risk of no-reflow during primary percutaneous coronary intervention (pPCI), while miR-126 can be used as a predictor of coronary slow flow phenomenon. In this study, we compared the diagnostic value of above genes in the prediction of no-reflow phenomenon in ST-segment elevation myocardial infarction (STEMI) subjects receiving pPCI. Quantitative real-time PCR, ELISA, Western blot and luciferase assays were performed to explore the regulatory relationship of MALAT1/miR-30e, MALAT1/miR-126, MALAT1/miR-155, miR-126/HPSE, and miR-155/EDN1. ROC analysis was carried out to evaluate the potential value of MALAT1, miRNAs and target genes in differentiating normal reflow and no-reflow in STEMI patients receiving pPCI. Elevated MALAT1, CRP, HPSE, and EDN1 expression and suppressed miR-30e, miR-155 and miR-126 expression was found in the plasma of STEMI patients receiving pPCI who were diagnosed with no-reflow phenomenon. ROC analysis showed that the expression of MALAT1, miR-30e, miR-126 and CRP could be used as predictive biomarkers to differentiate normal reflow and no-reflow in STEMI patients receiving pPCI. MALAT1 was found to suppress the expression of miR-30e, miR-126 and miR-155, and HPSE and EDN1 were respectively targeted by miR-126 and miR-155. This study demonstrated that MALAT1 could respectively sponge the expression of miR-30e, miR-126 and miR-155. And miR-30e, miR-126 and miR-155 respectively targeted CRP, HPSE and EDN1 negatively. Moreover, MALAT1 could function as an effective biomarker of no-reflow phenomenon in STEMI patients receiving pPCI.


Materials and methods
Human subjects and sample collection. In this study, we recruited 198 ST-segment elevation myocardial infarction (STEMI) patients receiving primary percutaneous coronary intervention (pPCI) during August 2018 to May 2021 in the department of cardiology at the Affiliated Hospital of Guilin Medical University. The patients were continuously enrolled and divided into two groups according to their reflow status as the normal reflow group (n = 152) and the no-reflow group (N = 46). No-reflow is diagnosed as the persistence of forward blood flow disorder [TIMI flow grade ≤ 2] after coronary artery without mechanical obstruction and no significant residual stenosis or dissection. The age, gender, diabetes history, hypertension history, hyperlipidemia history, familial history, smoking history, chest pain to hospitalization time, and door to balloon time, the angiography and procedural characteristics including infarct-related artery, multi-vessel disease, stent implantation and total stent length were collected and summarized for subsequent comparison. And serum samples were collected from all participants before their pPCI treatment. The ethical committee of Affiliated Hospital of Guilin Medical University has approved the protocol of this study (Approval ID: 2018-08-012). All procedures were performed in strict accordance with the last vision of the Declaration of Helsinki. Written informed consent was obtained from all patients or their first-degree relatives before the study.

RNA isolation and real-time PCR.
In this study, real-time PCR was done to compare the expression of MALAT1, miR-30e, miR-126, miR-155, CRP mRNA, HPSE mRNA, and EDN1 mRNA in each sample. In brief, total RNA in each sample was separated by utilizing a Trizol ® reagent (Invitrogen, Carlsbad, CA) following the standard assay procedure provided on the instruction manual of the supplier. In the next step, the quality and content of separated RNA was quantified at 260 nm and 280 nm absorbance by using a Nanodrop ND-3000 device (Thermo Fisher Scientific, Waltham, MA) following the standard assay procedure provided on the instruction manual of the instrument supplier. Then, 300 ng of separated total RNA from each sample were used to synthesis cDNA templates using a cDNA reverse transcription assay kit (Thermo Fisher Scientific, Waltham, MA) following the standard assay procedure provided on the instruction manual of the assay kit supplier. Finally, a Taqman Gene Expression Assay kit (Invitrogen, Carlsbad, CA) was used following the standard assay procedure provided on the instruction manual of the assay kit supplier to determine the relative expression of MALAT1, miR-30e, miR-126, miR-155, CRP mRNA, HPSE mRNA, and EDN1 mRNA in each sample via real time qPCR, which was carried out on a StepOnePlus (Invitrogen, Carlsbad, CA) real time qPCR machine following the standard assay procedure provided on the instruction manual of the instrument manufacturer. The Cell culture and transfection. HUVEC and HAEC cells were bought from Lonza (Walkersville, MD) and maintained following the standard incubation procedure provided by the supplier. In brief, the cells were maintained at 37 °C and 5% CO 2 in DMEM media (Gibco, Thermo Fisher Scientific, Waltham, MA) added with 10% FBS and penstrep. When HUVEC and HAEC cells reached confluency, they were divided into two groups, i.e., 1. NC siRNA group (HUVEC and HAEC cells transfected with negative control siRNA); and 2. MALAT1 siRNA group (HUVEC and HAEC cells transfected with MALAT1 siRNA). The transfection was carried out using Lipofectamine 3000 (Invitrogen, Carlsbad, CA) following the standard transfection procedure provided on the instruction manual of the transfection reagent supplier, and transfected cells were collected 48 h later to assay the expression of target genes.
Vector construction, mutagenesis, and luciferase assay. Our binding site screening results showed that miR-30e could potentially bind to MALAT1, so the luciferase vectors containing wild type and mutant promoter of MALAT1 carrying the miR-30e binding site were established and transfected into HUVEC and HAEC cells with miR-30e. In brief, the wild-type promoter sequence of MALAT1 carrying the miR-30e binding site was inserted into a pcDNA vector (Promega, Madison, WI) downstream the luciferase reporter gene to generate the wild type plasmid for the MALAT1. Then, a Quick Change III mutagenesis kit (Stratagene, San Diego, CA) was used following the standard assay procedure provided on the instruction manual of the assay kit supplier to induce a site-directed mutation in the miR-30e binding site of MALAT1 promoter, and the mutant sequence was also inserted into a pcDNA vector downstream the luciferase reporter gene to generate the mutant type plasmid for the MALAT1 promoter. In the next step, both luciferase vectors containing wild type and mutant MALAT1 were co-transfected into HUVEC and HAEC cells along with miR-30e, and the luciferase activity of transfected HUVEC and HAEC cells was assayed 48 h later using a Dual-Luciferase Report gene assay kit (Promega, Madison, WI) following the standard assay procedure provided on the instruction manual of the assay kit. Similarly, our binding site screening results showed that miR-126 could potentially bind to MALAT1, so the luciferase vectors containing wild type and mutant promoter of MALAT1 carrying the miR-126 binding site were established and transfected into HUVEC and HAEC cells with miR-126, according to the method similar to the one described above. In addition, to study the regulatory relationship of MALAT1/miR-155, miR-126/HPSE, and miR-155/EDN1, luciferase vectors containing wild type and mutant MALAT1, HPSE or EDN1 were established and co-transfected into HUVEC and HAEC cells along with miR-155, miR-126 and miR-155, respectively, and the luciferase activity of transfected HUVEC and HAEC cells was assayed 48 h later using the Dual-Luciferase Report gene assay kit. The reading of luciferase activity of transfected HUVEC and HAEC cells was done in a Turner luminometer (Turner Biosystems Luminometer, Promega, Madison, WI) following the standard assay procedure provided on the instruction manual of the luminometer.
Western blot analysis. The total protein from each sample was isolated first by using a RIPA lysis buffer (Sangon Biotech, Shanghai, China) following the standard assay procedure provided on the instruction manual of the lysis buffer supplier. In the next step, the concentration of isolated protein was quantified by utilizing a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Waltham, MA) following the standard assay procedure provided on the instruction manual of the assay kit supplier. Then, the total protein isolated from each sample was resolved on a 10% SDS-PAGE gel (Thermo Fisher Scientific, Waltham, MA) and blotted onto a PVDF membrane, which was then blotted in 5% skim milk and then incubated in sequence with primary anti-CRP, anti-HPSE, and anti-EDN1 antibodies as well as HRP-conjugated secondary antibodies (all antibodies were purchased from Santa Cruz biotechnology, Dallas, TX) following the standard incubation procedures provided on the instruction manual of the antibody supplier. Finally, after the PVDF membrane was developed in an enhanced chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA) following the standard assay procedure provided on the instruction manual of the reagent supplier, the protein bands were imaged and analyzed by using a chemiluminescence imaging system to calculate the relative protein expression of CRP, HPSE, and EDN1 in each sample.
ELISA. The plasma concentrations of CRP, HPSE, and EDN1 proteins in each patient were determined by utilizing corresponding enzyme-linked immunosorbent assay kits (R&D systems, Minneapolis, MN) following the standard assay procedure provided on the instruction manual of the assay kit supplier.

Statistical analysis. All statistical analyses were done by utilizing SPSS version 19 (SPSS, Chicago, IL) and
Prism version 7.0 (GraphPad, San Diego, CA). Differences with P < 0.05 were deemed statistically significant differences in this study. The comparison of non-continuous data between two or more groups was evaluated by using Chi square test. The comparison of continuous data between two and more groups was evaluated by using student t-test and one-way ANOVA, respectively. The normal distribution of each group was evaluated by using Kolmogorov-Smirnov analysis. Inter-group comparisons were carried out using Student's t-tests. The diagnostic value including both sensitivity and specificity was evaluated by using ROC analysis. The sample size was evaluated by using https:// www. stat. ubc. ca/ ~rollin/ stats/ ssize/ n2. html.

Results
Multivariate logistic regression analysis of reflow-related features of STEMI patients. As shown in Table 1, no significant differences were observed in respect to parameters including age, gender, diabetes history, hypertension history, hyperlipidemia history, familial history, smoking history, chest pain to hospitalization time, and door to balloon time between the normal reflow and no-reflow patient groups. Moreover, no remarkable difference was observed in respect to the angiography and procedural characteristics including infarct related artery, multi-vessel disease, stent implantation and total stent length were compared between the two groups (Table 2). However, the expression of MALAT1, miR-30e, miR-126, CRP, HPSE and EDN1 showed apparent differences between the two groups (Table 3).
The luciferase activities of MALAT1 were suppressed by miR-30e, miR-126 and miR-155. The luciferase activity of HPSE was inhibited by miR-126 and the luciferase activity of EDN1 was repressed by miR-155. Binding site screening respectively identified potential binding site of miR-30e ( Fig. 3A), miR-126 (Fig. 3B) and miR-155 (Fig. 3C) in MALAT1, and the luciferase activity of wild type MALAT1 was remarkably suppressed by the respective transfection of miR-30e (Fig. 3A), miR-126 (Fig. 3B) and miR-155     3C) in HUVEC and HAEC cells. Meanwhile, potential binding site of miR-126 was found in the 3′ UTR of HPSE, and luciferase activity of wild type HPSE was remarkably suppressed by miR-126 in HUVEC and HAEC cells (Fig. 3D). Also, miR-155 was found to potentially bind to EDN1, and the luciferase activity of wild type EDN1 was remarkably suppressed by miR-155 in HUVEC and HAEC cells (Fig. 3E).
MALAT1 siRNA activated the expression of miR-30e, miR-126 and miR-155, but suppressed the expression of CRP, HPSE and EDN1. The expression of MALAT1 was dramatically suppressed by MALAT1 siRNA in HUVEC and HAEC cells (Fig. 4A), but the expression of miR-30e (Fig. 4B), miR-126 (Fig. 4C) and miR-155 (Fig. 4D) was remarkably activated by MALAT1 siRNA in HUVEC and HAEC cells. On the contrary, the expression of CRP (Fig. 4E), HPSE (Fig. 4F) and EDN1 (Fig. 4G) mRNA was notably inhibited by MALAT1 siRNA in HUVEC and HAEC cells. Furthermore, the protein expression of CRP, HPSE and EDN1 was notably inhibited by MALAT1 siRNA in HUVEC and HAEC cells (Fig. 4H-K). The original blots of CRP, HPSE and EDN1 are present in the Supplementary Information.

Discussion
In this study, we recruited 198 ST-segment elevation myocardial infarction (STEMI) patients receiving primary percutaneous coronary intervention (pPCI) and divided them into two groups according to their reflow status. QPCR was performed to evaluate the expression of MALAT1, miR-30e, miR-126, and miR-155 in the plasma of patients with normal reflow and no-reflow. The expression of MALAT1 was remarkably enhanced, while the expression of miR-30e, miR-126 and miR-155 was significantly suppressed in the plasma of STEMI patients receiving pPCI who were diagnosed with no-reflow phenomenon. In addition, ELISA was performed to analyze the expression of CRP, HPSE and EDN1 in the plasma of patients with normal reflow and no-reflow. The expression of CRP, HPSE and EDN1 was notably upregulated in the plasma of STEMI patients receiving pPCI who were diagnosed with no-reflow phenomenon. MALAT1 was confirmed to be associated with numerous forms of tumors. For instance, MALAT1 is overexpressed in many cancer cells to control their proliferation, invasion, migration, and apoptosis 26 . Latest research also showed that lncRNA MALAT1 enhanced the invasion and proliferation of pancreatic, ovarian and glioma cancer cells by activating autophagy [27][28][29] . A previous research presented that MALAT1 regulated ATG5 expression   www.nature.com/scientificreports/ and autophagy through miR-30e 30 . Likewise, it was additionally discovered that miR-30e may be inhibited by MALAT1 to up-regulate ATG5 as well as activate autophagy in GC. Furthermore, MALAT1 contains two miR-126-5p miRNA response elements (MREs). Dual luciferase, biotin-RNA pull-down, as well as RNA-IP assays all presented that MALAT1 can sponge miR-126-5p 23 . MiR-155 was first identified to play an immune regulatory role in adaptive and innate immune responses 31,32 . It was also suggested that miR-155 plays an essential role in the onset of fibrosis 33,34 . Over-expressed in fibrotic tissues, miR-155 is essential to collagen metabolism by mediating TGF-1 signaling [35][36][37] . In this study, we used luciferase assay to explore the regulatory relationship of MALAT1/ miR-30e, MALAT1/miR-126, MALAT1/miR-155, miR-126/HPSE, and miR-155/EDN1. The luciferase activity of MALAT1 was effectively suppressed by miR-30e, miR-126 and miR-155. The luciferase activity of HPSE was inhibited by miR-126. The luciferase activity of EDN1 was repressed by miR-155. Moreover, we suppressed the expression of MALAT1 using MALAT1 siRNA. MALAT1 siRNA up-regulated the expression of miR-30e, miR-126 and miR-155, but suppressed the expression of CRP, HPSE and EDN1. It was show that pre-PCI plasma miRNA-30e expression in STEMI patients could be used to identify the risk of no-reflow. Furthermore, it was shown that the plasma levels of miRNA-30e were positively and significantly correlated with the level of LVEF upon admission, while negatively and considerably correlated with the expression level of hs-CRP. As a result of the rapid rise of CRP level in inflammatory conditions, CRP is considered as an important protein involved in the acute phase of inflammation by playing a significant role in mental stress, anxiety, neoplastic disease and myocardial infarction 2,38,39 .
Indicators like endothelin as well as CRP play an important regulatory role during the progression of noreflow 40 . In a recent study, the hs-CRP and ET-1 levels in the post-PCI peripheral blood were significantly elevated in the no-reflow group (P < 0.05). Moreover, the serum levels of hs-CRP showed the largest reduction within 3 h in the reflow group after PCI 41 . A number of mechanisms were proposed to support the hypothesis that high ET-1 levels may be used as a potential biomarker for the prediction of no-reflow phenomenon. As one of strongest vasoconstrictors in the body, ET-1 is produced and secreted from the vascular endothelia upon injury 42 . ET-1 also targets coronary arteries with low resistance. It has been revealed that the level of ET-1 was elevated in patients with ischemia ailments 43 . In patients with renal ischemia/reperfusion injury, the deletion of ET-1 from endothelial tissues protects the normal function of kidneys 44 . Moreover, ET-1 can additionally interact with polymorphonuclear leukocytes to induce no-reflow phenomenon by boosting the adherence of PMN leukocytes to endothelial tissues, therefore promoting the plugging of PMN 45 . Finally, ET-1 may enhance microvascular compression by elevating the permeability of microvasculature and by inducing edema 46 . Consequently, it is likely that the secretion of ET-1 in endothelia injured by ischemia may trigger sustained and rigorous microvascular constriction to potentiate no-reflow.
Heparanase is ubiquitously expressed to regulate angiogenesis, tumorigenesis, autophagy, fibrosis, and inflammation by cleaving HS groups from proteoglycans. The HS removal then lowers the stability of endothelial cell barriers while regulating the communication with cytokines, growth factors and chemokines 47 . The logistic regression revealed that miR-126 and also hs-CRP are independent risk factors of coronary slow flow. In addition, the plasma expression of miR-126 was significantly associated with the level of CSF, thus miR-126 may be utilized as a valuable predictor of coronary slow flow 48 . Elevated level of HPA in STEMI patients was associated with a high level of TB. Moreover, elevated level of HPA may predict thrombotic issues like no-reflow in STEMI patients 17 . In this study, we performed ROC analysis to evaluate the diagnostic potential of MALAT1, miR-30e, miR-126, miR-155, CRP, HPSE and EDN1 in differentiating normal reflow and no-reflow in STEMI patients receiving pPCI. MALAT1, miR-30e, miR-126 and CRP showed considerable efficiency in differentiating normal reflow and no-reflow in STEMI patients receiving pPCI.
The computational analysis indicated the MALAT1 as a regulator of miR-30e/CRP, miR-126/HPSE, and miR-155/END1. Furthermore, luciferase reporter assay results showed that there are interactions between MALAT1 and those three miRNAs (miR-30e, miR-126, and miR-155) and the CRP, HPSE, and END1 are the direct genes of miR-30e, miR-126, and miR-155, respectively. Those results were also confirmed by the results of cellular transfection of miRNA mimics. All those results that MALAT1 is a upstream regulator, which might be the basis for its role as a better biomarker for no-reflow phenomenon.

Conclusion
In this study, we demonstrated that MALAT1 could respectively sponge the expression of miR-30e, miR-126 and miR-155. The subsequent assays validated that miR-30e, miR-126 and miR-155 targeted CRP, HPSE and EDN1 in a negative manner. Therefore, since MALAT1 was found to present the highest AUC value among all studied genes, MALAT1 was shown to function as a biomarker of no-reflow phenomenon in STEMI patients who were subjected to pPCI.

Data availability
The data of this study are available from the corresponding author upon reasonable request.